Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Abstract

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM. Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells. The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

Physiological characterization of the wild almond Prunus arabica stem photosynthetic capability

“…For SEM analysis, stem samples fixed in formalin-aceticalcohol (FAA) (Bello et al, 2017) were passed through a series of ethanol solutions (50, 70, 90, 95, and 100%). The dehydrated samples were then dried in a quorum K850 critical point dryer (https://www.quorumtech.com) using liquid CO 2 .…”

Novel perspectives on stomatal impressions: Rapid and non-invasive surface characterization of plant leaves by scanning electron microscopy

“…Noninvasive sampling without inducing artefacts, demonstrated here, allows for repeated sampling of the same leaf. For other methods, including other leaf impression techniques, leaves must be detached to be imaged by SEM [6,8,12,20] making subsequent measurements of the same leaf impossible, and introducing nuisance inter-leaf variation to physiological studies. Replicas using this method allow for the creation of time-series with the same leaf, eliminating interleaf variations.…”

“…Scanning electron microscopy (SEM) is widely used to characterize the surface of plant leaves. It is a valuable tool to investigate the morphology of trichomes, stomatal density (SD), epicuticular waxes, accumulation of nano-particles, and infection with pathogens [1][2][3][4][5][6][7][8][9]. Biological samples for SEM have to be extensively prepared to withstand the vacuum and the high energy of the beam.…”

“…Sample preparation is usually invasive, irreversible, requires skill and expensive equipment, and is time and labor consuming. Leaves have to be cut off, sectioned into small pieces, fixed, dehydrated, critical point dried, and sputter coated before they can be investigated by SEM at room temperature [2,6,8]. Sample preparation can take several days and results in artifacts such as shrinking of sensitive structures [2,3,5,10,11].…”

Condiciones para establecer la relación phaseolus vulgaris - hongo entomopatógeno y su respuesta a estrés inducido por PEG

“…Tousimis®) con CO2 para lo cual las muestras se colocaron en cápsulas microporosas embebidas en alcohol al 100% e incorporando etiquetas de papel bond escritas a lápiz para identificarlas. Las muestras se montaron en soportes de aluminio y se recubrieron con una capa de oro de 500 Å de grosor en un recubridor de oro (BAL-TEC SDC 050) (Bozzola, et al, 1991); se observaron en un microscopio electrónico de barrido (JEOL modelo JSM-5900LV) y se obtuvo material micrográfico (Bello et al, 2017;Castillo-Minjarez, 2015;Barbosa-Martínez, 2003).…”